RESEARCH ARTICLE
483
Development 136, 483-493 (2009) doi:10.1242/dev.026955
EGFR signaling regulates the proliferation of Drosophila
adult midgut progenitors
Huaqi Jiang and Bruce A. Edgar*
In holometabolous insects, the adult appendages and internal organs form anew from larval progenitor cells during
metamorphosis. As described here, the adult Drosophila midgut, including intestinal stem cells (ISCs), develops from adult midgut
progenitor cells (AMPs) that proliferate during larval development in two phases. Dividing AMPs first disperse, but later proliferate
within distinct islands, forming large cell clusters that eventually fuse during metamorphosis to make the adult midgut epithelium.
We find that signaling through the EGFR/RAS/MAPK pathway is necessary and limiting for AMP proliferation. Midgut visceral
muscle produces a weak EGFR ligand, Vein, which is required for early AMP proliferation. Two stronger EGFR ligands, Spitz and
Keren, are expressed by the AMPs themselves and provide an additional, autocrine mitogenic stimulus to the AMPs during late
larval stages.
KEY WORDS: EGFR, Adult midgut progenitor (AMP), Intestinal stem cell (ISC)
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview
Ave N., Seattle, WA 98109, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 24 November 2008
undergoing several endocycles, reaching 64C (DNA content) by the
wandering L3 stage (Lamb, 1982). The AMPs remain diploid
throughout larval development and appear as scattered islets of cells
(hence the term ‘midgut histoblast islets’) in late-stage larval midguts.
During pupal development, the ECs histolyze and a new adult midgut
epithelium forms from the AMPs (Bender et al., 1997; Jiang et al.,
1997; Li and White, 2003). Similar midgut progenitor cells have also
been found in other insect species (Corley and Lavine, 2006).
Recently, the adult Drosophila midgut has been shown to undergo
dynamic self-renewal, a process similar to that found in the
mammalian intestine/colon. Fly and mammalian gut homeostasis
are both powered by intestinal stem cells (ISCs), and Notch
signaling plays similar roles in regulating their differentiation into
mature gut cells (Fre et al., 2005; Micchelli and Perrimon, 2006;
Ohlstein and Spradling, 2006; Ohlstein and Spradling, 2007; van Es
et al., 2005). Thus, the Drosophila midgut may serve as a model to
study gut homeostasis and the development of cancers, such as
colorectal carcinoma, that are directly associated with this dynamic
process in humans.
Here we describe the development of the AMPs in Drosophila
larvae and pupae. We discovered that Drosophila AMPs divide
extensively throughout larval development, and that their
proliferation can be separated into two distinct phases. During early
larval stages, the AMPs divide and disperse to form islets throughout
the midgut, but during late larval development the dividing AMPs
are contained within these islets. Furthermore, our study revealed
that Drosophila EGFR signaling is both necessary and sufficient to
induce the proliferation of AMPs during larval development.
MATERIALS AND METHODS
Fly stocks
UAS transgenes
The following were used: UAS-RasV12, UAS-RasV12S35, UAS-RasV12G37,
UAS-Rafgof, UAS-λTOP, UAS-SEM, UAS-RafDN, UAS-Mkp3, UAS-sSpi,
UAS-sKrn, UAS-Krn, UAS-grkΔTC and UAS-Vn1.2. UAS-RNAi transgenes
were obtained from the Bloomington Stock Center (Bloomington, IN,
USA), the National Institute of Genetics Fly Stock Center (NIG, Japan) or
the Vienna Drosophila RNAi Center (VDRC, Austria). According to
information from NIG and VDRC, all the RNAi lines used are specific to
the genes targeted (NIG, http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp;
VDRC, http://stockcenter.vdrc.at/control/main).
DEVELOPMENT
INTRODUCTION
The insect midgut, like the vertebrate intestine, is an endodermderived organ. Both the larval and adult Drosophila midguts are
composed of a single layer of epithelial cells with two layers of
visceral muscle (VM) wrapped outside. Inside the gut lumen, a
peritrophic membrane separates the food from the intestinal
epithelium. During both mammalian and insect embryonic
development, Forkhead and GATA transcription factors play
evolutionary conserved roles in the specification and subsequent
morphogenesis of the digestive tract (Stainier, 2005). Similarly,
multiple signaling pathways, including the EGF, Wingless (Wnt),
Dpp (TGFβ), Notch and Hedgehog pathways, are involved in the
embryonic development of the Drosophila midgut and mammalian
intestine (Sancho et al., 2004). In both systems, cross-talk between
mesodermal cells and endoderm-derived epithelial cells in the
gut primordium plays important roles during embryonic gut
development (Stainier, 2005; Szuts et al., 1998).
Starting from embryonic development stage 11, the Drosophila
midgut epithelium consists of two distinct cell populations:
differentiating midgut epithelial cells (larval enterocytes, ECs) and
undifferentiated adult midgut progenitors (AMPs, also referred to as
midgut histoblast islets or midgut imaginal islets) (Hartenstein et al.,
1992). In Drosophila embryos, AMPs can be marked by expression
of asense or by one of several lacZ- or Gal4-expressing enhancer-trap
insertions (Brand et al., 1993; Hartenstein et al., 1992; Hartenstein and
Jan, 1992). AMPs first appear as spindle-shaped cells localized to the
apical surface of the midgut epithelium, but later migrate to the basal
surface of the epithelium where they remain throughout larval
development (Hartenstein and Jan, 1992; Technau and CamposOrtega, 1986). Notch signaling has been shown to be involved in the
development of Drosophila AMPs. In Notch mutant embryos, the
number of AMPs in the midgut rudiment is strongly increased at the
expense of differentiated larval ECs (Hartenstein et al., 1992). During
larval development, the ECs grow in both size and ploidy by
RESEARCH ARTICLE
Mutants
FRT42D Egfrf1, FRT42D Egfr[CO], FRT82B Ras1Δc40b, spiA14 FRT40A,
FRT42D shot[65-2], FRT42D shot[V104], vnP1749 FRT80B, rhodel1 FRT80B,
Krn27-3-4, vnP1749, vnγ7, stet871 and ru1 were used (see FlyBase for further
information: http://flybase.org).
Gal4/lacZ reporters
esgGal4NP7397, spiGal4NP0261, MyoIAGal4NP0001 (NIG, Japan), rholacZAA69,
rholacZX81, howGal424B and esglacZK00606 were used (Bloomington Stock
Center).
Lineage analysis
MARCM lineage analysis
Newly hatched first instar [24 hours after egg deposition (AED)] or midthird instar (96 hours AED) larvae of the correct genotype were heat shocked
for 45 minutes at 37°C. The midguts were then dissected from wandering
L3 larvae (120 hours AED) and analyzed.
Flp/Gal4 lineage analysis
Newly hatched first instar larvae (24 hours AED) of the correct genotype
were heat shocked for 20 minutes at 37°C to induce clones and then
dissected at various developmental stages and analyzed.
Enhancer traps
P-element enhancer traps with midgut expression were obtained from
several sources, including FlyView (University of Münster, Germany;
http://flyview.uni-muenster.de) and GETDB (Gal4 Enhancer-Trap Insertion
Database, NIG, Japan). We identified a number of enhancer traps showing
reporter expression specifically in the AMPs, including one insertion in spi
(NP0261) and several insertions in esg (NP0726, 7397 and 7399).
esgGal4NP7397-driven GFP expression was used to mark the AMPs. We also
identified an enhancer trap in brush border Myosin IA (MyoIAGal4,
NP0001) that drives GFP expression specifically in midgut ECs (Morgan et
al., 1995).
Ectopic gene expression
We generated inducible AMP-, EC- and VM-specific expression systems
(esgGal4ts, MyoIAGal4ts and howGal4ts) by combining esgGal4NP7397,
MyoIAGal4NP0001 or howGal424B (Hartenstein and Jan, 1992) with
ubiquitously expressed temperature-sensitive alleles of the Gal4 inhibitor,
Gal80 (tubGal80ts; Bloomington Stock Center) and UAS-GFP.
Quantification of AMP clusters
We counted AMPs or AMP clusters marked by esgGal4-driven GFP
expression throughout the entire midgut during larval and pupal
development. UAS-GFP, UAS-sSpi, UAS-sKrn or UAS-Krn were induced
in the AMPs starting from first instar larvae (24 hours AED) using the
esgGal4ts system and the midguts were dissected from wandering L3 larvae
and the number of AMP clusters counted.
Generation of mutant AMP clones
Clones of AMPs homozygous for Egfrf1, Egfr[CO], Ras1Δc40b, spiA14, vnP1749,
rhodel1, shot[V104] or shot[65-2] were generated using the MARCM system
(Lee and Luo, 2001). First instar larvae (24 hours AED) of the correct
genotype were heat shocked for 45 minutes at 37°C to induce clones. Larvae
were then dissected at 120 hours AED. The number of GFP-positive clusters
in each clone was quantified; in most cases, clones from at least ten midguts
were counted.
RNA in situ hybridization and immunofluorescence
RNA in situ hybridization was performed as described (O’Neill and Bier,
1994). Rabbit anti-dpERK (Cell Signaling) was used to detect MAP
kinase activity in the midgut. Anti-Delta and anti-Prospero were obtained
from the Developmental Studies Hybridoma Bank and used to mark ISCs
and enteroendocrine cells in the midgut. Rabbit anti-β-galactosidase
(Cappel) was used to identify the esg-positive cells in an esglacZ
background. Rabbit anti-phospho-histone H3 (PH3, Upstate) was used to
identify dividing cells.
Development 136 (3)
Quantitative real-time PCR (qRT-PCR)
We used qRT-PCR to quantify levels of vn mRNA from midgut cDNA. For
mRpL30 (reference gene) primers see Buttitta et al. (Buttitta et al., 2007); for
vn: vn 5⬘ primer, 5⬘-TCACACATTTAGTGGTGGAAG-3⬘; vn 3⬘ primer, 5⬘TCACACATTTAGTGGTGGAAG-3⬘. The relative expression of vn was
analyzed on the Bio-Rad iQ5 system.
Sectioning
Wandering L3 midguts were dissected in PBS and fixed in half-strength
Karnovsky’s fixative. Following dehydration, the tissues were embedded in
Epon and sectioned at 1 μm. The sections were stained with Toluidine Blue.
RESULTS
Drosophila AMPs divide extensively during larval
development
To study the development of AMPs during larval development,
we first looked for AMP markers. From existing collections of
Drosophila enhancer traps we identified Gal4 or lacZ enhancer
traps that are expressed specifically in the AMPs. Among these
are Gal4 enhancer traps inserted in the escargot (esg) locus, which
encodes a member of the Snail family of transcription factors. esg
has previously been shown to be expressed in imaginal discs and
abdominal histoblast nests and is required there for maintaining
cells in the diploid state during larval development (Hayashi et al.,
1993). When combined with UAS-GFP, esgGal4 enhancer trap
NP7397 drove GFP expression specifically in the larval AMPs
(Fig. 1). Similar esg enhancer traps have been used to mark the
adult ISCs and their daughter enteroblasts (Micchelli and
Perrimon, 2006).
GFP expression driven by esgGal4 was detected in the AMPs
scattered throughout the midgut of newly hatched larvae (24 hours
AED) (Fig. 1A, arrows). AMPs appeared as small diploid cells, and
were easily distinguishable from the large polyploid midgut
enterocytes (ECs). The number of GFP-positive AMPs increased
during early larval development (24-72 hours AED) (Fig. 1A,B);
however, they remained dispersed. Cell contacts between paired
AMPs were readily observed in the early larval midgut (Fig. 1B,
inset) and are likely to represent two daughter AMPs from the
previous division migrating away from each other. By mid-third
instar (96 hours AED), AMPs formed discrete 2- to 3-cell clusters
(Fig. 1C), suggesting that they proliferate within individual islets
instead of migrating away from each other. The AMPs continued to
proliferate within these clusters (Fig. 1D), undergoing several
rounds of rapid proliferation to enlarge each cluster to 8-30 cells by
the onset of metamorphosis [0 hours after pupae formation (APF),
~130 hours AED] (Fig. 1E).
These results do not support the idea that Drosophila AMPs are
quiescent during larval development (Bodenstein, 1994). Instead,
we observed that the AMPs proliferate extensively during larval
development, resulting in large increases in both the number
(early larval stages) and size (late larval stages) of the AMP
clusters. To further document this process, we analyzed AMP
lineages by positively marking individual AMPs with GFP using
the MARCM system (Lee and Luo, 2001). When clones were
induced in first or second instar (24-48 hours AED), they all
contained multiple AMP clusters by the wandering L3 stage (120
hours AED), and all cells in any GFP-positive cluster were GFPpositive (Fig. 2A-A⬙; Fig. 4A). However, when clones were
induced in mid-third instar (96 hours AED), clusters mosaic for
GFP were observed by the wandering L3 stage (120 hours AED)
(Fig. 2B-B⬙). These results confirmed that the AMPs switch to
proliferating within islets to form clusters by mid-third instar. To
quantify the number of divisions during the early proliferative
DEVELOPMENT
484
Drosophila AMP development
RESEARCH ARTICLE
485
phase, we counted the number of the marked AMP clusters
encompassed by each clone. When induced in the newly hatched
first instar larvae (24 hours AED), the clones contained, on
average, 7.5 GFP-positive clusters at the wandering L3 stage (120
hours AED) (see Table S1 in the supplementary material). This
suggests that the AMPs divide about four times during the early
larval stages (note that only half of all the clusters generated by
each AMP were marked in the MARCM system). Since no mosaic
AMP clusters were found in the late larval midgut when clones
were induced at first or second instar, we propose that the
majority, if not all, of the early larval AMPs disperse after each
cell division. We then counted the number of cells in each cluster
at white prepupa formation (0 hours APF), when most of the AMP
clusters have stopped proliferating. Each AMP cluster contained
8 to greater than 30 cells. This indicates that the AMPs divide an
additional three to five times within a cluster, after the clusters are
established. In total, the AMPs appear to divide seven to ten times
throughout larval development.
Midgut development during early metamorphosis
Staining for the division marker phospho-histone H3 (PH3)
indicated that by white prepupa formation (0 hours APF), the
majority of the AMPs had ceased their proliferation. Some AMPs in
the posterior midgut, however, did not cease proliferation until 4
hours APF (data not shown). Meanwhile, the visceral muscles
(VMs) contracted, and the larval midgut shortened itself
dramatically. At the same time, the AMP clusters fused to form a
new midgut epithelium (Fig. 1F), while the larval midgut epithelium
became extremely compacted and was sloughed into the intestinal
lumen (Li and White, 2003). The midgut continued to contract and
shorten, and by 12 hours APF it became a sac-like structure
containing histolyzing larval epithelium inside the newly formed
midgut (Juhasz and Neufeld, 2008). Visceral muscles undergo a
process termed ‘de-differentiation’, in which the muscle fibers
histolyze; however, the muscle cells themselves do not die and will
redifferentiate to form the adult midgut VM during late
metamorphosis (Klapper, 2000). During early metamorphosis (0-24
DEVELOPMENT
Fig. 1. Development of Drosophila adult midgut progenitors (AMPs). AMPs were marked by GFP expression (green) driven by esgGal4NP7397.
The numbers of GFP-positive AMPs, AMP clusters or adult intestinal stem cells (ISCs) are indicated in the appropriate panels. DNA is stained with
DAPI (blue). (A) First instar larval midgut (24 hours AED). GFP was detected in the AMPs as individual diploid cells (arrows). (B) Early third instar larval
midgut (72 hours AED). Larval enterocytes (ECs) undergo several rounds of endoreplication, enlarging the larval midgut. AMPs remain diploid and
their numbers increase during the first two larval stages. However, they remain mostly dispersed as individual cells. Inset shows GFP expression that
is overexposed to show cell contacts between two neighboring AMPs. (C) Mid-third instar larval midgut (96 hours AED). AMPs form distinctive 2- to
3-cell clusters. (D) Late third instar larval midgut (120 hours AED). AMPs continue to proliferate and enlarge the clusters. (E) White prepupa stage (0
hours APF, ~130 hours AED). The size of the AMP clusters has increased further. (F) Prepupa stage (4 hours APF). The AMP clusters fuse to form a
new midgut epithelium. Larval ECs (out of focal plane) are sloughed into the lumen and histolyze. (G,H) Early pupa stage (8 and 12 hours APF). The
majority of the cells in the new midgut epithelium gradually lose GFP expression, except for a few scattered cells that maintain strong GFP
expression. (I,J) Pupa stage (24 hours APF). The future adult ISCs are clearly identifiable by strong GFP expression (asterisks in I) and basal
localization in the epithelium (asterisks in J, cross-sectional view). GFP expression is lost in the rest of the cells in the new epithelium. Scale bars:
20 μm.
486
RESEARCH ARTICLE
Development 136 (3)
Fig. 2. Lineage analysis of the AMPs.
(A-B⬙) Drosophila AMP clones induced using the
MARCM system. Clones were induced at either first
instar (24 hours AED, A-A⬙) or third instar (96 hours
AED, B-B⬙) and analyzed at the wandering L3 stage
(120 hours AED). When induced at first instar, the
clones appear as multiple marked clusters with all cells
labeled (A-A⬙), whereas clones induced late (at 96 hours
AED) were all confined to a single cluster that is mosaic
for GFP (B-B⬙). (C-E⬙) Pupal or adult AMP clones induced
using the Flp/Gal4 system. Flp/Gal4 AMP clones were
induced at first instar larval stage (24 hours AED) and
analyzed at 24 hours APF (C-C⬙) or from newly eclosed
adults (D-E⬙). At 24 hours APF, each AMP clone contains
0-2 esg-positive cells (C, arrows); the asterisk marks the
histolyzing larval midgut. In newly eclosed adults, the
midgut contains enteroendocrine cells and ISCs; arrows
indicate cells within the clone that are positive for
Prospero (D) and Delta (E).
smaller, basally localized cells positive for the enteroendocrine cell
marker Prospero or the ISC marker Delta (Fig. 2D-D⬙,E-E⬙). These
results indicate that some of the AMPs become adult ISCs
(Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006), and
that this transition occurs in the pupa. This was further supported by
our observation that AMP clones induced in the larva persisted in
the adult midgut for at least 2 months (data not shown).
EGFR signaling stimulates AMP proliferation
Using the esgGal4ts system, we manipulated the activity of several
known Drosophila signaling pathways specifically in the AMPs.
Our tests included Wingless, Dpp, Hedgehog, Notch and EGFR
signaling components (see Table S2 in the supplementary material).
Activation of EGFR/RAS/MAPK signaling in the AMPs was able
to drive their overproliferation during larval development.
Compared with control midguts (Fig. 3A), in which the AMPs
appeared as 2- to 3-cell clusters by mid-third instar (96 hours AED),
the induction of activated Ras (Ras oncogene at 85D – FlyBase)
(RasV12) in the AMPs led to the formation of much larger AMP
clusters (Fig. 3B-D). Ectopic expression of RasV12 in the AMPs
DEVELOPMENT
hours APF), the majority of cells in the newly formed midgut
epithelium gradually lost esgGal4-driven GFP expression, with the
exception of a few scattered cells that maintained strong GFP
expression (Fig. 1F-I, asterisks). By 24 hours APF, these GFPpositive cells became basally localized in the new epithelium (Fig.
1J, asterisks). As described below, we believe that these cells are the
future adult midgut ISCs (see Discussion).
Around 24 hours APF, the esg-positive cells in the new adult
midgut epithelium started to proliferate (see Fig. S1A-A⵮ in the
supplementary material). They continued to divide at 48 hours APF
and increased in number (see Fig. S1B-B⵮ in the supplementary
material). At 72 hours APF, the esg-positive cells continued to
divide and some of them also expressed the enteroendocrine cell
marker Prospero (see Fig. S1C-C⵮ in the supplementary material),
indicating their capacity to differentiate. Whether these cells already
behaved as stem cells, which both self-renew and differentiate, was
not determined. GFP-marked AMP Flp/Gal4 clones induced at early
larval stages contained only 0-2 esg-positive cells at 24 hours APF
(Fig. 2C-C⬙), but when these clones were scored later, in newly
eclosed adults, they contained both large apically localized ECs and
Drosophila AMP development
RESEARCH ARTICLE
487
supplementary material), whereas expression of RasV12G37, which
preferentially activates the Phosphotidylinositol 3 kinase (PI3K) or
Ral guanine nucleotide exchange factor 2 (RalGDS) pathway
(Karim and Rubin, 1998; Prober and Edgar, 2002), had little effect
on their proliferation (see Table S2 in the supplementary material).
Second, ectopic expression of Dp110 (Pi3K92E – FlyBase; PI3K)
had no detectable effect on AMP proliferation (see Table S2 in the
supplementary material). Third, increased proliferation of the AMPs
was observed when activated Egfr (λTOP) (Queenan et al., 1997),
gain-of-function Raf (Rafgof) (Brand and Perrimon, 1994) or
activated MAPK [sevenmaker (sem); rolled – FlyBase] (MartinBlanco, 1998) was induced in these cells (see Table S2 in the
supplementary material). Fourth, expression of a dominant-negative
form of Raf (RafDN) (Roch et al., 1998) together with RasV12 gave a
phenotype similar to that of RafDN alone (see Fig. S2E,F in the
supplementary material), and thus Raf is epistatic to Ras in
regulating AMP proliferation. Fifth, expression of Mkp3, a negative
regulator of MAPK (Rintelen et al., 2003), did not affect AMP
proliferation (see Fig. S2G in the supplementary material).
Interestingly, however, Mkp3 did significantly suppress the AMP
overproliferation phenotype induced by RasV12 expression (see Fig.
S2H in the supplementary material; compare with Fig. 3D).
throughout larval development resulted, by the wandering stage
(120 hours AED), in a midgut comprising mostly esg-positive
AMPs (Fig. 3F) in which the intestinal lumen was occluded. By
contrast, wild-type AMPs appeared as basally localized cell clusters
in the midgut epithelium (Fig. 3E). The following evidence
suggested that EGFR signaling promoted AMP proliferation
through activating the MAPK pathway. First, induction of RasV12S35,
which preferentially activates the MAPK pathway, drove similar
ectopic proliferation of the AMPs as did RasV12 (see Table S2 in the
Drosophila MAPK is activated in the AMPs
To examine whether downstream components of EGFR signaling
are activated in the AMPs, we stained the larval midgut with
antibodies against diphospho-extracellular signal-regulated kinase
(dpERK; Rolled – FlyBase), the level of which is a direct
measurement of the activated form of Drosophila MAPK (Gabay et
al., 1997). dpERK staining was indeed detected in the AMP clusters,
but not in the larval gut epithelial cells (Fig. 5A-A⬙), indicating
activation of MAPK in these cells. This result is consistent with our
genetic results and supports the notion that EGFR signaling
regulates AMP proliferation through activating the MAPK pathway.
DEVELOPMENT
Fig. 3. Activated Ras (RasV12) stimulates AMP proliferation. UAStransgenes were induced in the Drosophila AMPs using the esgGal4ts
system. Larvae were shifted to 29°C at the indicated times and
dissected at 96 hours AED. (A) GFP (24-96 hours AED, control).
(B) RasV12 (72-96 hours AED). (C) RasV12 (48-96 hours AED). (D) RasV12
(24-96 hours AED). (E,F) Cross-sections of posterior midguts from
wandering L3 larvae expressing ectopic GFP (E, wild type, WT) or RasV12
(F) throughout larval development (24-120 hours AED). The control
AMP clusters are basally localized in the epithelium (E, arrows).
PM, peritrophic membrane. The samples in E and F were stained with
Toluidine Blue.
EGFR signaling is required for AMP proliferation
Next, we tested whether the EGFR/RAS pathway is required for the
normal proliferation of AMPs during larval development. Using the
same esgGal4ts system, we depleted crucial components of the
pathway by expressing RNA inverted repeats (IR, RNAi) specific to
Drosophila Egfr, Ras and Raf (pole hole – FlyBase) in the AMPs.
As a control, UAS-driven RNAi directed against GFP was induced
in the AMPs using the esgGal4ts system. This treatment did not
affect AMP development (data not shown). When compared with
control midguts from white prepupa (0 hours APF), RNAi-mediated
depletion of each of these gene products in the AMPs throughout
larval development significantly decreased both the number and size
of the AMP clusters (see Fig. S2A-D and Table S2 in the
supplementary material). This indicates that both phases of AMP
proliferation were affected when EGFR signaling was
downregulated. Similar results were observed when the dominantnegative form of Raf (RafDN) was induced in the AMPs (see Fig.
S2E and Table S2 in the supplementary material).
In further tests we generated AMP clones defective in EGFR
signaling using the MARCM system. AMPs mutant for Egfr
(Egfr[CO]) or Ras (Ras1Δc40b) did not proliferate during larval
development (Fig. 4; see Table S1 in the supplementary material).
Instead of forming multiple GFP-positive clusters as in controls
(Fig. 4A), these mutant clones appeared as single GFP-positive cells
(Fig. 4B,C). We conclude that EGFR/RAS/MAPK signaling is
required for AMP proliferation during both early and late larval
development.
RESEARCH ARTICLE
Fig. 4. EGFR signaling mutant AMPs fail to proliferate. Egfr–/– or
Ras–/– Drosophila AMP clones were generated using the MARCM
system, which positively marks mutant cells with GFP expression.
(A) FRT42D only (control). (B) FRT42D Egfr[CO]. (C) FRT82B RasΔc40b. The
boxed regions in A-C are shown to the right as GFP (A⬘-C⬘), DNA
(A⬙-C⬙) and merged (A⵮-C⵮) images. Unlike in the control (A-A⵮), where
GFP-positive clones form multiple clusters, clones of Egfr[CO] and
RasΔc40b AMPs (B-B⵮; C-C⵮) appear as single GFP-positive cells.
Arrowheads indicate the positions of Egfr–/– or Ras–/– AMPs. The asterisk
in C indicates one larval EC with non-specific GFP expression from the
MARCM system (FRT82B).
Next, we examined the expression patterns of several EGFR
ligands in the larval midgut. We identified a Gal4 enhancer trap
in spi (NP0261, see Materials and methods) that drove UAS-GFP
expression specifically in the AMPs (Fig. 5B-B⬙). RNA in situ
hybridization confirmed that spi was specifically expressed in the
AMP clusters (Fig. 5D,D⬘). Krn was also specifically expressed
in the AMPs as shown by RNA in situ hybridization (Fig. 5E,E⬘).
Development 136 (3)
Multiple EGFR ligands are involved in AMP
proliferation
To investigate which EGFR ligands regulate the proliferation of the
AMPs, we expressed each of the four known EGFR activating
ligands (gurken, spitz, Keren and vein) in the AMPs using the
esgGal4ts system. Induction of activated gurken (grkΔTC), a strong
EGFR ligand, the function of which is believed to be exclusively in
female oogenesis (Nilson and Schupbach, 1999), did not affect the
proliferation of the AMPs (see Table S2 in the supplementary
material). However, induction of activated (secreted) spitz or Keren
(sSpi or sKrn), two other strong EGFR activating ligands (Reich and
Shilo, 2002; Schweitzer et al., 1995), promoted extensive
overproliferation of the AMPs (Fig. 6A-C; see Table S2 in the
supplementary material). Furthermore, induction of wild-type Krn,
which requires cleavage by rhomboid family proteases to become
fully active, similarly promoted AMP proliferation (Fig. 6D-D⬘; see
Table S2 in the supplementary material). In addition, induction of
sSpi or sKrn limited the dispersal of the AMPs, thus reducing the
number of the clusters in the wandering L3 larval midguts (Fig. 6E).
Induction of the weak EGFR ligand vein (vn) (Schnepp et al., 1998)
with esgGal4ts, however, had little effect on AMP proliferation (see
Table S2 in the supplementary material).
To determine whether spi or Krn are required for AMP
proliferation, we downregulated the levels of these EGFR ligands in
the AMPs by RNAi. Ectopic expression of UAS-RNAi directed at spi
and/or Krn using esgGal4 had no effect on AMP proliferation (see
Table S2 in the supplementary material). Consistent with this, the
proliferation of the AMPs in Krn27-3-4 (null allele) mutant larvae was
normal (see Table S1 in the supplementary material). The same was
also found for spiA14 mutant AMP clones generated in a Krn27-3-4
homozygous mutant background (see Table S1 in the supplementary
material). The proliferation of mutant AMPs lacking rhomboid
(rhodel1, null allele) or spi (spiA14, null allele) function, generated
using the MARCM system, was also normal (see Table S1 in the
supplementary material). We examined lacZ expression from two
rholacZ reporters in the larval midgut (rhoAA69 and rhoX81) and found
that neither were expressed in the AMPs. Since the Drosophila
genome encodes multiple rhomboid-like genes, we also generated
MARCM clones in the mutant background of rho-2 (stet871) and
rho-3 (ru1). These mutant clones also contained normal numbers of
AMP clusters (see Table S1 in the supplementary material). These
results suggest that either multiple, redundant rhomboid-like genes
are utilized in the AMPs, or (less likely) that rhomboid-like function
is dispensable in the larval midgut. Furthermore, we conclude that
spi and Krn are likely to be dispensable for AMP proliferation (see
Discussion).
Surprisingly, in several vn mutants (vnP1749/P1749, vnγ7/γ7 and
vnP1749/γ7), few AMP clusters were found in the late larval midgut (Fig.
7B,C), whereas the AMPs in wild-type controls formed many large
clusters (Fig. 7A). This suggests that vn is required for normal AMP
development. To further study the function of vn in AMP
development, we carried out lineage analysis of the AMPs in the vn
mutant animals. We induced GFP-marked AMP clones in first instar
larvae using the Flp/Gal4 system. Compared with control midguts,
which contained on average 15.2 marked AMP clusters per clone
(n=50 clones) (see Fig. S3A-A⬙ in the supplementary material), we
consistently observed only a single GFP-positive AMP cluster in the
midguts of weak vn mutants (vnγ7/P1749; animals of this genotype are
not developmentally delayed during larval development and most die
as pharate adults) (see Fig. S3B-B⬙ in the supplementary material).
Furthermore, we counted the number of esg-positive cells (marked by
esglacZ) in newly hatched larval midguts. Control larval midguts
DEVELOPMENT
488
Drosophila AMP development
RESEARCH ARTICLE
489
(vnP1749/+) contained on average 121 AMPs per gut, whereas there
were on average 137 AMPs per midgut in vnγ7/P1749 mutants (ten
midguts for each genotype were scored). This indicates that the
reduction in the number of AMP clusters in the late larval midgut of
vnγ7/P1749 mutants was not due to the production of fewer AMPs
during embryogenesis. Taken together, these results suggest that the
proliferation of AMPs during the early larval stages is completely
inhibited in vn mutants. However, the size of the few remaining AMP
clusters in the vnγ7/P1749 mutant midguts was relatively normal (see
Fig. S3B-B⬙ in the supplementary material), suggesting that the late
phase of AMP proliferation is largely unaffected in vn mutants. We
speculate that the reason vn becomes dispensable for AMP
proliferation during late larval development is that Krn and spi
expression in the AMPs supplies a redundant function.
Interestingly, we found that vn is specifically expressed in VM
cells throughout larval development, as revealed by the expression
of a well-characterized lacZ enhancer-trap insertion, vnlacZP1749
(Fig. 5C-C⬙) (Kiger et al., 2000). The Drosophila midgut VM
comprises an outer layer of 21 longitudinal muscle strips and an
inner layer of circular muscle that forms four distinctive rows
(Klapper, 2000), as revealed by a muscle-specific driver,
howGal424B, which drives UAS-GFP expression in both types of
VM cells (Fig. 5C⬘) (Brand and Perrimon, 1993). vnlacZ expression
was stronger in the inner, circular VM cells than in the outer,
longitudinal muscle (Fig. 5C).
To test the importance of Vn signaling from VM, we specifically
depleted vn from VM cells by expressing UAS-Vn RNAi using an
inducible muscle-specific driver, howGal4ts. Induction of vn RNAi
throughout larval development (24-120 hours AED) resulted in late
larval midguts with very few AMP clusters, as in vn mutants (Fig.
7D-D⬙). However, induction of vn RNAi starting at early third instar
(72 hours AED) had no effect on the AMPs (Fig. 7E-E⬙).
Furthermore, induction of UAS-Vn RNAi in the AMPs or in the
larval ECs using the esgGal4ts or MyoIAGal4ts (MyoIA is also
known as Myo31DF – FlyBase) drivers had no effect on AMP
proliferation (see Fig. S4A,B in the supplementary material),
suggesting that the principal source of Vn is VM. This was
confirmed by quantitative real-time PCR showing that induction of
UAS-Vn RNAi in VM significantly reduced vn mRNA levels in
whole midguts, whereas induction of vn RNAi in ECs or AMPs did
not (Fig. 7G). In further tests, we attempted to rescue the AMP
phenotype of vnP1749 mutants by expressing UAS-Vn in AMPs, ECs
or VM, using the esgGal4ts, MyoIAGal4ts or howGal4ts drivers.
Induction of UAS-Vn in the AMPs or VM completely rescued the
phenotype of vnP1749 mutants (Fig. 7F-F⬙; see Fig. S5A-A⬙ in the
supplementary material). Induction of vn in the larval ECs, which
constitute the bulk of the midgut mass, not only rescued the
proliferative defects of the AMPs, but also caused ectopic AMP
proliferation (see Fig. S5B-B⬙ in the supplementary material). We
conclude that Vn, expressed in VM, is the principal mitogen for
AMPs during early larval development. Later, autocrine Spi and Krn
might complement this function.
This scenario, in which VM-derived Vn activates EGFR signaling
in AMPs, is reminiscent of the role of Vn in muscle/tendon
development during embryogenesis. In this case, muscle-derived Vn
is specifically concentrated on tendon cells and activates EGFR
DEVELOPMENT
Fig. 5. Expression and activity of the EGFR
ligands spitz, Keren and vein in the larval
midgut. (A-A⬙) MAPK activity (dpERK staining) in
the midgut of late third instar Drosophila larvae.
(B-B⬙) The expression of UAS-GFP driven by
spiGal4NP0261 in the midgut of L3 wandering larvae.
(C-C⬙) vnlacZ reporter expression pattern in the
midgut. Large arrows indicate the circular visceral
muscle cells, which form four distinct rows (two are
shown). Small arrows indicate the longitudinal
visceral muscle cells. (D,D⬘) Krn RNA in situ
hybridization in L3 wandering larval midgut.
(E,E⬘) spi RNA in situ hybridization in L3 wandering
larval midgut. Arrowheads indicate the positions of
the AMP clusters in all panels.
490
RESEARCH ARTICLE
Development 136 (3)
Fig. 6. Expression of sSpi, Krn or sKrn in the AMPs induces their
proliferation. The ligands were induced in the Drosophila AMPs using
the esgGal4ts system starting at 24 hours AED, and larvae were
dissected at 96 hours AED. (A,A⬘) GFP (control). (B,B⬘) Activated
(secreted) Spi (sSpi). (C,C⬘) Activated (secreted) Krn (sKrn). (D,D⬘) Krn.
(A-D) GFP marks the AMP clusters. (A⬘-D⬘) Merged images of GFP
(green) and DNA (DAPI, blue). (E) The ectopic expression of strong EGF
ligands in the AMPs dramatically reduces the total number of AMP
clusters in the midgut. WT, wild-type.
there (Strumpf and Volk, 1998). The concentration of Vn is highly
dependent upon the activity of the short stop (shot, also called
kakapo) gene in the tendon cells (Strumpf and Volk, 1998). We
tested whether shot is also required for VM-derived Vn to activate
DISCUSSION
Drosophila AMPs undergo extensive proliferation
during larval development
Drosophila AMPs were previously thought to be relatively
quiescent during larval development, dividing just once or twice,
and not initiating rapid proliferation until the onset of
metamorphosis (Bodenstein, 1994). This is the case for several
other larval progenitor/imaginal cell types, such as the abdominal
histoblasts and cells in the salivary gland, foregut and hindgut
imaginal rings (Bodenstein, 1994). More recent studies have
suggested that AMP proliferation might precede the onset of
metamorphosis (Hall and Thummel, 1998; Jiang et al., 1997; Li and
White, 2003). However, these studies did not report the extensive
proliferation of the AMPs that we describe here, and failed to
recognize the early larval proliferative phase when the AMPs
divide and disperse (Figs 1 and 8). The extensive proliferation of
the AMPs is similar to that of the larval imaginal disc cells, which
also proliferate throughout larval development, dividing about ten
times.
Lineage analysis revealed that the proliferation of the Drosophila
AMPs occurs in two distinct phases (Fig. 8). In early larvae, the
AMPs divide and disperse throughout the midgut to form individual
islets. During later larval development, the AMPs continue to divide
but do so within these islets, forming large cell clusters. We
speculate that in the early larva, secretion of Vn from the midgut
visceral muscle (VM) cells results in low-level activation of EGFR
signaling in the AMPs, which is sufficient for their proliferation and
might also promote their dispersal. We did not observe any
proliferation defects in AMPs defective in shot function, suggesting
that the mechanism of EGFR activation used by tendon cells during
muscle/tendon development is probably not the same as in the larval
midgut. Specifically, it is unlikely that the Shot-mediated
concentration of Vn on AMPs activates EGFR signaling in the
AMPs during early larval development. Consistent with this, we
only observed dpERK staining in AMP clusters (Fig. 5A-A⬙) and
not in the isolated AMPs present at early larval stages (24-72 hours
AED; data not shown).
The mechanisms that regulate the transition between these two
proliferation phases remain unclear. We observed fewer AMP
clusters when sSpi, sKrn, λTOP (activated Egfr) or RasV12 were
induced in the AMPs starting from early larval stages (Fig. 6E; see
Table S2 in the supplementary material), suggesting that EGFR
signaling, in addition to its crucial role as an AMP mitogen, might
also play a role in AMP cluster formation. In the late larval midgut
(96-120 hours AED), high-level EGFR activation, resulting from
expression of spi and Krn in the AMPs themselves, might not only
promote AMP proliferation, but might also suppress AMP
dispersal and thus promote formation of the AMP clusters. How
the timing and location of Spi- or Krn-mediated EGFR activation
are regulated during larval development is also unclear. We note,
however, that the pro-ligand form of Krn acted similarly to sKrn
(Fig. 6), and that we failed to uncover any functions for the Rholike gene products that regulate Spi and Krn function by
proteolytic cleavage in other tissues (see Tables S1 and S2 in the
supplementary material). This suggests that the localized
expression of these ligands in the AMP clusters might be the
DEVELOPMENT
EGFR signaling in the AMPs by quantifying AMP clusters in shot
mutant MARCM clones. These clones all contained normal
numbers of AMP clusters (see Table S1 in the supplementary
material), and thus the role of shot in transducing the Vn signal is
uncertain.
Drosophila AMP development
RESEARCH ARTICLE
491
critical parameter that controls their effects. Consistent with this,
Rho-independent cleavage and function of Krn have been
documented (Reich and Shilo, 2002).
In the developing Drosophila wing, EGFR/RAS/MAPK
signaling promotes the expression and controls the localization of
the cell adhesion molecule Shotgun (Shg, Drosophila DE-cadherin)
(O’Keefe et al., 2007). RasV12-expressing clones generated in the
wing imaginal disc are round (Prober and Edgar, 2002), much like
the AMP clusters described here, owing to increased adhesive
junctions. In developing Drosophila trachea, EGFR activity
upregulates shg expression to maintain epithelial integrity in the
elongating tracheal tubes (Cela and Llimargas, 2006). In the eye,
EGFR activity leads to increased levels of Shg and adhesion
between photoreceptors (Brown et al., 2006; Mirkovic and
Mlodzik, 2006). Given these precedents, it seems reasonable to
suggest that high-level EGFR activity in the AMP islets upregulates
Shg and promotes the homotypic adhesion of the AMPs.
Alternatively, changes in the differentiated cells of the midgut
epithelium might promote AMP clustering. In either case, the
dispersal of early AMPs and subsequent formation of late AMP
clusters facilitate the formation of the adult midgut epithelium
during metamorphosis.
AMPs give rise to adult intestinal stem cells
during metamorphosis
Our study confirms previous reports that Drosophila AMPs replace
larval midgut epithelial cells to form the adult midgut epithelium
during metamorphosis (Figs 1 and 8) (Bender et al., 1997; Jiang et al.,
1997; Li and White, 2003). Furthermore, we show that the majority
of AMPs lose esgGal4-driven GFP expression as they differentiate to
form the new adult midgut epithelium (Fig. 1F-J). These cells lacked
Prospero, which marks enteroendocrine cells in both the larval and
adult midgut (Micchelli and Perrimon, 2006; Ohlstein and Spradling,
2006). They went through several rounds of endoreplication during
late pupal development (not shown), and thus probably all
differentiated into adult enterocytes (ECs). During early
metamorphosis, some cells in the new midgut epithelium remained
small and diploid and maintained strong esgGal4 expression (Fig.
1I,J; Fig. 8). For several reasons, we believe that these esg-positive
cells are the future adult intestinal stem cells (ISCs). First, esgGal4
expression marks AMPs, including adult ISCs and enteroblasts
(Micchelli and Perrimon, 2006). Second, mitoses in the adult midgut
are only observed in ISCs (Micchelli and Perrimon, 2006; Ohlstein
and Spradling, 2006), and we observed mitoses only in the esgpositive cells during metamorphosis (see Fig. S1 in the supplementary
DEVELOPMENT
Fig. 7. Vein is required for AMP proliferation.
(A-C) Posterior midguts from white prepupa (0 hours APF) of
wild-type (WT) Drosophila contain multiple AMP clusters (A),
which are missing from the midguts of vn mutants (B, vnP1749;
C, vnγ7). Midguts are outlined with dashed lines.
(D-E⬘) Posterior midguts from wandering L3 larvae in which
vn was specifically knocked down in the visceral muscle cells
throughout larval development (24-120 hours AED, D) or only
during late larval development (72-120 hours AED, E). Most
of the remaining small cells in B-D are visceral muscle cells.
Arrows in D point to the few AMP clusters in the midgut.
(F,F⬘) Induction of UAS-Vn expression throughout larval
development (24-120 hours AED) using the muscle-specific
driver howGal4ts rescued the vn mutant phenotype. D⬘-F⬘
show merged images of DNA (DAPI, blue) and howGal4tsdriven GFP expression (green) in the visceral muscle and
trachea (asterisks in D⬘,F⬘) cells. (G) Knockdown of vn mRNA
in the midgut by vn RNAi. Relative levels of vn mRNA in the
larval midgut were quantified by qRT-PCR. Only UAS-Vn RNAi
expression driven by the muscle-specific Gal4 driver,
howGal4ts, knocked down vn significantly in the larval
midgut.
492
RESEARCH ARTICLE
Development 136 (3)
Fig. 8. Postembryonic development of the
Drosophila midgut epithelium. AMPs (green)
proliferate in two phases and several EGFR ligands
are involved in each phase. Also note the
specification of future adult intestinal stem cells
during early metamorphosis and the reappearance of
enteroendocrine cells (red) at a late stage of
metamorphosis (72 hours APF). See text for details.
Implications for EGFR/RAS signaling in insect
midgut development
EGFR signaling is both required and sufficient to promote AMP
proliferation (Figs 3, 4, 6 and 7; see Fig. S2 in the supplementary
material). Hyperactivation of EGFR signaling, such as by expression
of activated Ras (RasV12), promoted massive AMP overproliferation
and generated hyperplastic midguts that were clearly dysfunctional
(Fig. 3F). On the other hand, inhibiting EGFR/RAS/MAPK
signaling dramatically reduced AMP proliferation (Fig. 4; see Fig.
S2 in the supplementary material). Furthermore, the ability of EGFR
signaling to induce ectopic AMP proliferation is almost unique.
With the exception of larval hemocytes (Zettervall et al., 2004),
activated EGFR signaling does not promote cell proliferation in the
imaginal discs, salivary gland imaginal rings, abdominal histoblasts,
foregut and hindgut imaginal rings. This suggests that the regulation
of AMP proliferation is different from that in other imaginal cells.
Regulation of AMP proliferation by non-epithelial
muscle cells
Despite the obvious differences between adult ISCs and their
larval progenitors, the AMPs, there are also similarities. First,
when the new adult midgut epithelium forms, larval AMPs give
rise to the new adult midgut including the adult ISCs. Many
genes, such as esg, that are specifically expressed in the larval
AMPs are also expressed in the adult ISCs (our unpublished data).
Second, the structure of the midgut epithelium with basal AMPs
or ISCs is similar in larval and adult stages. Third, vn expression
in larval VM persists in the adult midgut (our unpublished data),
suggesting that Vn from the adult VM might also regulate the
ISCs.
In two Drosophila stem cell models, the testis and ovary, stem
cells reside in special niches comprising other supporting cell types.
These niches maintain the stem cells and provide them with
proliferative cues (Ohlstein et al., 2004). For example, in the testis,
germ stem cells attach to the niche that comprises cap cells. The cap
cells release Jak/Stat and BMP ligands [Upd (Os) and Gbb/Dpp],
which maintain the stem cells and induce their proliferation.
Whether Drosophila ISCs utilize supporting cells that constitute a
niche remains unclear. Here we show that multiple EGFR ligands
are involved in the regulation of Drosophila AMP proliferation.
During early larval development, the midgut VM expresses the
EGFR ligand vn (Fig. 5C-C⬙), which is required for AMP
proliferation (Fig. 7; see Fig. S3B-B⬙ in the supplementary
material). Thus, the early AMPs might be considered to require a
niche comprising non-epithelial VM. Later in larval development,
however, the AMPs express two other EGFR ligands, spi and Krn
(Fig. 5E,F), which are capable of autonomously promoting their
proliferation (Fig. 6) and may render vn dispensable (Fig. 7E,E⬘; see
Fig. S3B-B⬙ in the supplementary material). We found, however,
that depleting spi and Krn in the AMPs did not affect AMP
proliferation, suggesting that vn or another trigger of
EGFR/RAS/MAPK activity might complement spi and Krn in latestage larvae.
DEVELOPMENT
material). Third, esg-positive cells migrated to the basal side of the
midgut epithelium (Fig. 1J), the location of adult ISCs (Micchelli and
Perrimon, 2006; Ohlstein and Spradling, 2006). Fourth, AMP clones
generated during early larval development contained just a few esgpositive cells when the new adult midgut first formed (24 hours APF)
(see Fig. S1C-C⬙ in the supplementary material), but when such clones
were scored in newly eclosed adults, they contained large numbers of
ECs, as well as cells positive for the enteroendocrine marker Prospero
and the ISC marker Delta (Fig. 2D,E). This suggests that a small
fraction of AMPs differentiate into adult ISCs. However, esg-positive
cells in the new pupal midgut lacked Delta expression until eclosion
(Fig. 2E-E⵮; data not shown), suggesting that they are probably not
mature adult ISCs.
How a small fraction of AMPs are selected to become adult ISCs
in the newly formed pupal midgut epithelium is not known. One
possibility is that the adult ISCs are determined during larval
development, long before the formation of the adult midgut. Another
is that they are specified during early metamorphosis. We prefer this
second hypothesis for several reasons. First, in our lineage analysis,
we found that all AMP clones induced during early larval stages
formed multiple clusters (Fig. 2A-A⬙; see Fig. S3A-A⬙ in the
supplementary material). This suggests that there are no quiescent
AMPs in the larval midgut. Second, when AMP clones were induced
at mid-third instar, the mosaic clusters always contained multiple
GFP-positive cells, suggesting that all AMPs in the mid-third instar
midgut remain equally proliferative (Fig. 2B-B⬙). Third, during
larval development, we never observed differentiation of the AMPs,
as judged by their ploidy (diploid) and lack of expression of the
enteroendocrine marker Prospero (not shown). Fourth, all AMPs
appeared to express esgGal4 throughout larval development. Given
the crucial role that Notch signaling plays in regulating AMPs
during embryonic midgut development (Hartenstein et al., 1992) and
ISCs in adult midgut homeostasis (Micchelli and Perrimon, 2006;
Ohlstein and Spradling, 2006; Ohlstein and Spradling, 2007), we
expect that Notch might also function to specify adult ISCs during
metamorphosis.
We thank Amanda Simcox, Matthew Freeman, Jocelyn McDonald, Ryu Ueda,
Celeste Berg, Laura Johnston, Andrew Dingwall, Margaret Fuller and the NIG
(Japan), VDRC (Vienna) and Bloomington (USA) Stock Centers for providing
flies; the FHCRC EM Laboratory for preparing larval midgut sections; the
Moen’s lab for their help with confocal imaging; two anonymous reviewers for
their helpful comments; and members of Edgar lab for their support, especially
Dr Tao Wang, Dr Parthive Patel and Aida de la Cruz for critical reading of the
manuscript. This work was supported by pilot funds from the UW/FHCRC
Cancer Consortium and NIH grant R01 GM51186 to B.A.E. Deposited in PMC
for release after 12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/3/483/DC1
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DEVELOPMENT
Drosophila AMP development